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Journal of Virology, May 1999, p. 4413-4426, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparative Analysis of Evolutionary Mechanisms of the
Hemagglutinin and Three Internal Protein Genes of Influenza B
Virus: Multiple Cocirculating Lineages and Frequent Reassortment of
the NP, M, and NS Genes
Stephen E.
Lindstrom,
Yasuaki
Hiromoto,
Hidekazu
Nishimura,
Takehiko
Saito,
Reiko
Nerome, and
Kuniaki
Nerome*
Department of Virology I, National Institute
of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan
Received 9 December 1998/Accepted 9 February 1999
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ABSTRACT |
Phylogenetic profiles of the genes coding for the hemagglutinin
(HA) protein, nucleoprotein (NP), matrix (M) protein, and nonstructural
(NS) proteins of influenza B viruses isolated from 1940 to 1998 were
analyzed in a parallel manner in order to understand the evolutionary
mechanisms of these viruses. Unlike human influenza A (H3N2) viruses,
the evolutionary pathways of all four genes of recent influenza B
viruses revealed similar patterns of genetic divergence into two major
lineages. Although evolutionary rates of the HA, NP, M, and NS genes of
influenza B viruses were estimated to be generally lower than those of
human influenza A viruses, genes of influenza B viruses demonstrated
complex phylogenetic patterns, indicating alternative mechanisms for
generation of virus variability. Topologies of the evolutionary trees
of each gene were determined to be quite distinct from one another,
showing that these genes were evolving in an independent manner.
Furthermore, variable topologies were apparently the result of frequent
genetic exchange among cocirculating epidemic viruses. Evolutionary
analysis done in the present study provided further evidence for
cocirculation of multiple lineages as well as sequestering and
reemergence of phylogenetic lineages of the internal genes. In
addition, comparison of deduced amino acid sequences revealed a novel
amino acid deletion in the HA1 domain of the HA protein of recent
isolates from 1998 belonging to the B/Yamagata/16/88-like lineage. It
thus became apparent that, despite lower evolutionary rates, influenza
B viruses were able to generate genetic diversity among circulating
viruses through a combination of evolutionary mechanisms involving
cocirculating lineages and genetic reassortment by which new variants
with distinct gene constellations emerged.
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INTRODUCTION |
Influenza type A and B viruses share
many characteristics both biologically and biochemically. For example,
both viruses possess a segmented genome consisting of eight
negative-strand RNA segments which encode three polymerase proteins
associated with polymerase activities, a hemagglutinin glycoprotein
(HA), a neuraminidase glycoprotein (NA), a nucleoprotein (NP), a matrix
protein (M1), and two nonstructural proteins (NS1 and NS2). However,
there are also significant differences between the epidemiology,
evolutionary patterns, and host ranges of influenza A and influenza B
viruses. In fact, influenza A viruses are subdivided into 15 HA and 9 NA subtypes, all of which have been isolated from aquatic avian species and many of which also infect other avian and mammalian species, including humans, horses, mink, whales, swine, and seals (11, 15,
22, 30, 36). The segmented genome of influenza viruses allows
different influenza viruses to exchange gene segments during coinfection of a cell (reassortment). Through genetic reassortment, pandemic influenza viruses with novel hemagglutinin subtypes (antigenic shift) which are able to evade established immunity may suddenly appear
in humans. Antigenic shift has occurred at least twice in the twentieth
century, both times resulting in major pandemics causing high morbidity
and mortality in humans around the world. In 1957, the emergence of
H2N2 (Asian) virus was the result of reassortment between avian H2N2
and human H1N1 (Spanish) virus. Again in 1968, antigenic shift occurred
as a result of reassortment between H2N2 (Asian) virus and an avian H3
virus to create the human H3N2 (Hong Kong) virus. In contrast,
influenza B viruses, which have been isolated only from humans, consist
of a single HA and NA type and thus are incapable of antigenic shift.
Furthermore, due to high evolutionary rates of influenza A viruses, the
HA and NA proteins are able to evade established immunity in humans by
gradually changing their antigenic profile (antigenic drift). By this
mechanism, the HA gene of human influenza H3N2 viruses has evolved in
essentially a sequential manner to generate new antigenic strains which
displace variants of the previous season (8-10, 27).
Although the HA protein of influenza B viruses undergoes antigenic
drift, evolution of the HA protein of influenza B viruses has been
characterized by a lower rate of antigenic change and cocirculation of
antigenic variants for considerable periods of time (20, 39,
46). In particular, since the mid-1980s, the HA gene of influenza
B viruses has been shown to have evolved into two distinct lineages
represented by B/Yamagata/16/88- and B/Victoria/2/87-like viruses and
to have demonstrated a mechanism of systematic insertion and deletion
of nucleotides (20, 21, 32, 39). It was recently observed
that outbreaks of influenza B virus in Japan tend to occur in
association with H3N2 viruses, with influenza B virus activity peaking
after that of H3N2 virus, usually in March (32).
Nevertheless, it is still unclear how, despite slow evolutionary change
and the lack of antigenic shift, influenza B viruses are able to
continue to cause seasonal epidemic episodes in humans.
In addition to differences in host range specificity and evolutionary
patterns of the HA genes, influenza A and B viruses show further
differences in their protein coding strategies. RNA segment 6 of
influenza A virus is monocistronic, coding for the NA protein, while
segment 6 of influenza B virus is bicistronic, possessing two
overlapping open reading frames (ORFs) which code for the NA and NB
proteins. The NB protein of influenza B viruses is a membrane protein
which is believed to serve a function similar to that of the M2 protein
of influenza A viruses (1, 2, 42, 44). Furthermore, although
RNA segment 7 of both influenza A and B viruses codes for the M1
(matrix) protein, the organizations of their respective M2 genes are
quite different. Processing of the M2 gene of influenza A viruses
involves posttranscriptional splicing of the mRNA to obtain the M2 ORF.
The M2 protein subsequently shares the first 14 amino acids (aa) with
the M1 protein, while the remaining 88 aa are unique to the M2 protein
(17, 24). On the other hand, RNA segment 7 of influenza B
viruses is bicistronic, containing tandem cistrons characterized by
overlapping of the termination codon of the M1 gene and the initiation
codon of the BM2 gene, resulting in a rare coupled
termination-initiation mechanism of viral gene expression
(16). Although the NB protein of influenza B viruses is
believed to form a membrane ion channel similar to that of the M2
protein of influenza A viruses (42, 44), influenza A viruses
have no counterpart for the M2 protein of influenza B viruses (BM2),
whose function remains unknown.
Although the phylogenetic patterns of the HA genes of influenza B
viruses are well understood, evolutionary characteristics of the
internal genes have not been well resolved. Recent analysis of seven
influenza B virus NP genes provided evidence for multiple lineages and
reassortment (19), while Yamashita et al. (46) suggested multiple lineages of the NS gene. Recent advances in molecular techniques, such as rapid RNA purification, reverse transcription (RT)-PCR, direct sequencing of PCR amplicons, and automated nonradioisotopic DNA sequencing, have allowed for rapid and
safe analysis of a large number of viral genes. In this report, we
compare the phylogenetic profiles of the HA, NP, M, and NS genes of 20 influenza B viruses from 1940 to 1998 in a parallel fashion in order to
better understand the evolutionary mechanisms of these viruses. A
comparison of evolutionary mechanisms of influenza A and B viruses is
also discussed.
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MATERIALS AND METHODS |
Viruses.
The following influenza B virus strains whose genes
were sequenced in this study were propagated in 11-day-old embryonated chicken eggs: B/Bangkok/64, B/Osaka/70, B/Gifu/73, B/Guma/73, B/Kanagawa/3/76, B/Norway/4/84, B/Ibaraki/2/85, B/Victoria/2/87, B/Aichi/5/88, B/Yamagata/16/88, B/Panama/45/90, B/Mie/1/93,
B/Guangdong/8/93, B/Beijing/184/93, B/Guangdong/5/94, B/Harbin/7/94,
B/Argentina/218/97, B/Beijing/243/97, B/Henan/22/97, B/Nara/4/97,
B/Hiroshima/97/97, B/Shiga/44/98, B/Chiba/447/98, B/Nagano/2038/98,
B/Shiga/51/98, B/Shiga/T30/98, and B/Yamanashi/166/98.
RNA extraction and nucleotide sequencing.
Viral RNA was
extracted from 100 µl of virus sample by use of a commercial kit
(RNeasy; Qiagen) and suspended in a final volume of 40 µl of
RNase-free distilled water. RT-PCR was performed by using a modified
protocol of a commercial kit (RT-PCR kit with avian amyeloblastosis
virus reverse transcriptase, version 2.1; Takara) as described
previously (26). RT reactions were done with 20 pmol of a
previously described universal influenza B virus RT primer (5'
AGCAGAAGC 3') (47) and 9.5 µl of RNA sample/20 µl
of RT reaction mixture. Aliquots of 5 µl of the resultant cDNA were
then used in all subsequent PCR amplifications of overlapping cassettes
covering the entire protein coding domain of the NP, M, and NS genes,
as well as the HA1 domain of the HA gene, by using the following
thermocycler program: initial denaturation at 94°C for 2 min followed
by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C
for 1 min and a final extension at 72°C for 10 min. Resultant
amplicons were purified and sequenced directly by methods described
previously (41) on an ABI377 or ABI310 autosequencer
(Perkin-Elmer). Oligonucleotide primers for PCRs and sequencing
reactions were designed to have annealing temperatures ranging from 62 to 66°C. Nucleotide sequences for all oligonucleotide primers used
for PCR amplification and sequencing are available from the authors
upon request.
Phylogenetic analyses and evolutionary rates.
Phylogenetic
trees were constructed by using the neighbor-joining method (12,
31, 40) and bootstrap analysis (n = 500) to
determine the best-fitting tree for each gene. Nucleotide distance matrices were estimated by the six-parameter-method (12)
based on the number of total nucleotide substitutions, and evolutionary trees for the HA, NP, M, and NS genes were constructed (see Fig. 1 to
4, respectively) (13, 40). In addition to the sequence data
determined in this study, previously reported nucleotide sequences for
the HA gene (20, 21, 23, 32, 38, 39, 45, 46), NP gene
(5, 7, 19, 28, 35, 37), M gene (4, 7, 14), and NS
gene (3, 33, 46) were also used in the construction of
phylogenetic trees. The abbreviations used for influenza B virus
strains are as follows: B/Lee/40 (Lee40), B/Bon/43 (Bon43), B/Great
Lakes/54 (GL54), B/Maryland/59 (Mar59), B/Singapore/64 (Sin64), B/Ann
Arbor/66 (Ann66), B/Russia/69 (Rus69), B/Hong Kong/8/73 (HK73),
B/Yamagata/73 (Yam73), B/Baylor/4/78 (Bay78), B/Singapore/222/79
(Sin79), B/Paris/1/79 (Par79), B/Fukuoka/80/81 (Fuk81),
B/England/222/82 (Eng82), B/Houston/1851/84 (Hou84), B/Georgia/1/86
(Geo86), B/Idaho/1/86 (Ida86), B/Ann Arbor/1/86 (Ann86),
B/Nagasaki/1/87 (Nag87), B/Beijing/1/87 (Bei87), B/USSR/2/87 (USSR87),
B/Shanghai/2/87 (Sha87), B/Finland/56/88 (Fin88), B/Taiwan/7/88 (Tai88), B/Singapore/7/88 (Sin88), B/India/3/89 (Ind89),
B/Victoria/19/89 (Vic1989), B/Victoria/103/89 (Vic10389), B/South
Dakota/5/89 (Sou89), B/Guangdong/55/89 (Gua89), B/Paris/329/90 (Par90),
B/Finland/151/90 (Fin90), B/New York/3/90 (NY90), B/Texas/4/90 (Tex90),
B/Bangkok/163/90 (Ban90), B/Hong Kong/22/89 (HK2289), B/Hong Kong/9/89
(HK989), B/Texas/1/91 (Tex91), B/Osaka/1/93 (Osa93), B/Tokyo/101/93
(Tok93), B/Mie/1/93 (Mie93), B/Kagoshima/15/94 (Kag94), B/Kobe/1/94
(Kob94), B/Hebei/19/94 (Heb1994), B/Hebei/3/94 (Heb394), B/Harbin/7/94 (Har94), B/Yamagata/74/94 (Yam94), B/Sendai/240/95 (Sen24095), B/Sendai/243/95 (Sen24395), B/Sendai/136/95 (Sen13695), B/Tokyo/942/96 (Tok96), B/Sapporo/1/96 (Sap96), B/Nagasaki/1/96 (Nag96),
B/Alaska/12/96 (Ala96), B/Osaka/491/97 (Osa97). The remaining strain
abbreviations are indicated in Table 1.
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TABLE 1.
Accession numbers of influenza B virus sequence data
determined in this study and abbreviations of the virus isolates
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Evolutionary rates based on the total number of nucleotide
substitutions in the HA, NP, M, and NS genes as well as the number
of
amino acid substitutions in the HA, NP, M1, BM2, NS1, and NS2
proteins
were calculated by plotting the evolutionary distance
(number of
substitutions per site) of each virus from a putative
origin against
the year of isolation and determining the slope
of the best-fit line by
regression
analysis.
Nucleotide sequence accession numbers.
Nucleotide sequence
data determined in this study will appear in the GSDB, DDBJ, EMBL, and
NCBI nucleotide sequence databases under the accession numbers listed
in Table 1.
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RESULTS |
Analysis of the HA gene.
Analysis of the HA gene of 77 influenza B viruses (Fig. 1) was
undertaken in order to provide a basis for comparison of evolutionary patterns of the internal genes, as well as to understand the
phylogenetic location of the HA genes of recent isolates from 1997 to
1998. In support of previous analyses (20, 21, 32, 38, 39), the HA gene was demonstrated to have evolved into three major lineages.
Lineage I included earliest isolates from 1940 until the mid-1970s,
while more recent isolates were found to form two distinct lineages
which appeared to have diverged prior to the isolation of Sin79. These
results support previous reports describing cocirculation of
antigenically and phylogenetically distinct Yamagata/16/88-like (lineage II) and Victoria/2/87-like (lineage III) variants (20, 21, 32, 38, 39). Phylogenetic divergence of lineages II and III
was supported by calculated bootstrap probabilities of 95 and 100%,
respectively. Analysis of recent influenza B virus isolates from 1997 to 1998 revealed that variants of both lineage II and lineage III were
isolated in Japan in 1998, forming new clades at the ends of each
lineage. Also, viruses of both lineages were isolated from the same
geographic region; Shi4498 and Shi5198 belonged to lineage III, while a
third virus isolated from the same area, Shi3098, was included in
lineage II.
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FIG. 1.
Evolutionary tree based on the total number of
nucleotide substitutions of the HA1 domain of the HA gene of human
influenza B viruses constructed by neighbor-joining analysis. Isolates
indicated in blue represent viruses belonging to lineage II, whereas
those indicated in red represent viruses belonging to lineage III.
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A comparison of predicted amino acid sequences of the HA1 domain of the
HA proteins of recent isolates to that of HK73 (Table
2) revealed many
conserved changes which were characteristic
of each lineage. However,
it was interesting to observe that as
many as 12 aa changes occurred
independently at the same positions
in viruses of both lineages. For
example, even though phylogenetically
distinct viruses Shi5198 (lineage
III) and Shi3098 (lineage II)
differed by 32 aa (9.2%), 11 of these
changes were at similar
locations. Independent changes at similar sites
may be indicative
of lower functional constraints on amino acids at
these positions
or, possibly, escape from immune pressure directed to
these sites.
However, the latter argument does not explain why viruses
of both
lineages demonstrated similar amino acid changes at positions
88 (K to R [K-R]), 129 (T-K, lineage III; R-K, lineage II), and
267 (V-I). The HA protein of influenza B viruses is characterized
by the
insertion and deletion of amino acids at positions 163
to 165 (
20,
32,
39). It was, therefore, relevant that three
of the four most
recent viruses of lineage II, Arg97, Shi3098,
and YN98, were found to
have a novel deletion at position 162.
Estimation of nucleotide and amino acid substitution rates of the HA1
domain of the HA gene of influenza B viruses (Table
3) revealed that the evolutionary rates
of both lineages calculated
here were somewhat lower than those
previously reported. Nucleotide
substitution rates for lineages II and
III were estimated to be
2.41 × 10
3 nucleotide
substitutions/site/year (ns/st/yr) and 1.39 × 10
3
ns/st/yr, respectively, which are the converse of previously
described
rates of 3.42 × 10
3 and 4.17 × 10
3 ns/st/yr, respectively (
39). These
differences were likely
attributable to inclusion of more sequence data
collected over
a longer period of time in the calculation of this
report. It
was also apparent that the HA1 domain of the HA gene of
influenza
B viruses was evolving at a rate about three times lower than
that of influenza A H3N2 viruses (5.7 × 10
3
ns/st/yr) (
9). At the amino acid level, the HA protein of
lineage II was observed to be evolving at a rate of 3.36 × 10
3 amino acid substitutions/site/year (aas/st/yr), which
was slightly
higher than that of lineage III (2.18 × 10
3 aas/st/yr) and was consistent with the amino acid
variability
observed in this study and previously (
38).
Analysis of the NP gene.
Phylogenetic analysis of the NP gene
of 24 influenza B viruses isolated since 1940 revealed evolutionary
profiles similar to those of the HA gene, showing that the NP gene has
evolved into three major lineages (Fig.
2). Lineage I was represented by the
classical isolates Lee40 and Ann66, while more recent viruses were
observed to have diverged into two distinct lineages (lineages II and
III). However, unlike the evolutionary patterns of the HA gene, NP
genes of lineage III appeared to have circulated for a relatively short
period of time, since this lineage consisted entirely of viruses
isolated between 1984 and 1988. Nevertheless, lineages II and III were
demonstrated to have bootstrap confidence levels of 99 and 100%,
respectively. Also, as can be seen in Fig. 2, the constituent viruses
of each lineage contrasted considerably with those of the HA gene. For
example, the NP genes of viruses of HA lineage III and HA lineage II
were not restricted to either NP lineage. Rather, NP lineages II and
III consisted of a mixture of viruses from both HA lineage II and HA
lineage III, reflecting frequent reassortment of these genes among
influenza B viruses. These results confirm evidence for reassortment
among influenza B viruses based on the evolutionary position of the NP
gene of Tex88 observed by Jambrina et al. (19). Further
analysis of the NP gene in this study demonstrated that reassortment
appeared to have occurred quite regularly among influenza B viruses,
including the most recent isolates from 1998. Interestingly, all NP
genes of recent viruses belonged to lineage II, forming two branch
clusters, IIi and IIii, representing viruses of HA lineage II and HA
lineage III, respectively.
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FIG. 2.
Evolutionary tree of the NP gene of human influenza B
viruses based on the total number of nucleotide substitutions in the
complete protein coding region constructed by neighbor-joining
analysis. Isolates indicated in red represent viruses whose HA gene
belonged to lineage I, while isolates indicated in blue represent those
whose HA gene belonged to lineage II.
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Alignment of deduced amino acid sequences of the NP proteins of both
lineages with that of Ann66 (Table
4)
revealed lower
variability in the NP protein when compared to the HA
protein.
There were a total of 12 conserved (i.e., shared by three or
more
viruses) aa differences (2.1%) between the NP proteins of lineage
II and lineage III, while the NP proteins of recent isolates of
branch
clusters IIi and IIii differed by only 4 conserved aa (0.7%)
at
positions 17 (T-A, lineage IIii), 171 (I-V, lineage IIii),
233 (L-I,
lineage IIi) and 520 (K-R, lineage IIi).
The evolutionary rate of the NP gene of influenza B viruses (Table
3)
of lineage II (0.95 × 10
3 ns/st/yr) was
approximately one-fourth that previously estimated
(3.6 × 10
3 ns/st/yr) (
37). Also, this rate was about
one-half that of
the NP gene of influenza A viruses (2.3 × 10
3 ns/st/yr), while the NP protein of influenza B
viruses (0.26
× 10
3 aas/st/yr) was calculated to be
evolving at a rate which was
almost 1/10 that of influenza A viruses
(2.1 × 10
3 aas/st/yr) (
43).
Analysis of the M gene.
Construction of the evolutionary tree
of the M gene of influenza B viruses included the complete protein
coding region of 1,076 bp comprising the M1 and M2 ORFs of the M gene
of 24 viruses (Fig. 3). The M gene was
found to have evolved into three distinct lineages which, with the
exception of one virus, were topologically identical to those of the NP
gene, with lineages II and III having bootstrap confidence values of 96 and 100%, respectively. Similar to the NP gene, the M genes of most
recent viruses were all located in lineage II, forming two branch
clusters, IIi and IIii, whose HA genes belonged to HA lineages II and
III, respectively. However, it was interesting to note that the M gene
of Gua93 was phylogenetically located in lineage III, indicating a
reemergence of the M gene of lineage III after a 9-year hiatus. This
was in contrast to the NP gene of this virus, which was included in
lineage II of the evolutionary tree of the NP gene.
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FIG. 3.
Evolutionary tree of the M gene of human influenza B
viruses based on the total number of nucleotide substitutions in the
complete protein coding region constructed by neighbor-joining
analysis. Isolates indicated in red represent viruses whose HA gene
belonged to lineage I, while isolates indicated in blue represent those
whose HA gene belonged to lineage II.
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A comparison of predicted amino acid sequences of the M proteins of
influenza B viruses (Table
5) revealed
almost complete
conservation of the M1 proteins among influenza B
viruses when
compared with that of Ann66. Regardless of phylogenetic
distinctions,
there were no conserved amino acid differences found in
the M1
proteins of viruses isolated during a 32-year period from 1966
to 1998. Similar to the conserved nature of the M1 protein of
influenza
A viruses (
18,
26), the lack of variability in the
M1
protein of influenza B viruses suggested high functional constraints
on
this protein. Although the function of the BM2 protein is not
yet well
understood, all viruses contained a conserved BM2 ORF
of 330 nucleotides coding for a predicted polypeptide of 109 aa,
supporting
the BM2 ORF described by Horvath et al. (
16). In
contrast to
the M1 protein, high amino acid variability was demonstrated
among BM2
proteins. Eleven conserved amino acid differences (10.1%)
were
observed between lineages II and III, while most recent isolates
varied
by five conserved amino acids (4.6%).
Lineages II and III of the M gene of influenza B viruses demonstrated
similar nucleotide substitution rates of 1.09 × 10
3
and 1.31 × 10
3 ns/st/yr (Table
3), respectively,
which were comparable to that
calculated for human A viruses (1.08 × 10
3 ns/st/yr) (
18). Nucleotide sequences of
the M1 ORF of B viruses
of lineage II displayed conservative rates of
0.75 × 10
3 (lineage II) and 0.30 × 10
3 (lineage III) ns/st/yr, while evolutionary rates of
the BM2 ORF
of lineages II and III (2.01 × 10
3 and
4.14 × 10
3 ns/st/yr, respectively), on the other
hand, were markedly higher.
In addition, the rates of change of the BM2
ORF of influenza B
viruses were higher than that of the M2 coding
region of influenza
A viruses (1.36 × 10
3
ns/st/yr). At the protein level, the M1 protein of influenza
B viruses
displayed virtually no evolution, since there were no
conserved amino
acid changes observed among virus isolates from
1966 to 1998. In
contrast, the BM2 protein of lineages II and
III was observed to be
evolving at rates of 3.80 × 10
3 and 3.46 × 10
3 aas/st/yr, respectively, which were unexpectedly
higher than
those of the HA protein. Furthermore, these rates were
about 2.5
times higher than that of the M2 protein of human A viruses
(1.38
× 10
3 aas/st/yr) (
18), although it
should be noted that the M2 protein
of influenza A viruses and the BM2
protein of influenza B viruses
are not believed to share
functionality.
Analysis of the NS gene.
Calculation of the phylogenetic tree
of the NS genes of influenza B viruses included 32 sequences determined
in this report and previously (3, 33, 46) and revealed
divergence into four major lineages (Fig.
4). Lineage I included the NS genes of
the earliest isolates, Lee40 and GL54, with those of later viruses of
the 1950s and 1960s being located in lineage II. Similar to the
evolution of the NP and M genes, the NS gene of more recent viruses was
characterized by phylogenetic divergence into two major lineages,
supporting analysis by Yamashita et al. (46), who described
multiple lineages of the NS gene based on the location of the NS gene
of Hou84. Although the NS genes of recent isolates were found to be
divided into two major lineages, evolutionary distances of NS genes of
each lineage were often not proportional to their years of isolation,
suggesting cocirculation of minor sublineages. Moreover, analysis of
this report further revealed that viruses containing HA genes of HA
lineages II and III were distributed throughout both NS gene lineages
III and IV and that the general topology of the NS tree was quite
distinct from those of the NP and M genes. Thus, the NS gene of
influenza B viruses demonstrated a pattern of genetic reassortment
which was distinct from those of the NP or M genes. In particular, the
NS gene of the representative strain of HA lineage III, Vic87, was
located in NS lineage III together with that of Yam88, the
representative strain of HA lineage II. Also, in contrast to the NP and
M genes of isolates from 1997 and 1998, the NS genes of all isolates
from 1997 and 1998 were located in lineage IV, forming minor clades, IVi and IVii, representing viruses of HA lineages III and II, respectively. Regardless, lineages III and IV appeared to have diverged
prior to the isolation of HK73 with respective bootstrap probability
values of 85 and 99%.
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FIG. 4.
Evolutionary tree of the NS gene of human influenza B
viruses based on the total number of nucleotide substitutions in the
complete protein coding region constructed by neighbor-joining
analysis. Isolates indicated in red represent viruses whose HA gene
belonged to lineage I, while isolates indicated in blue represent those
whose HA gene belonged to lineage II.
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A considerable number of conserved amino acid changes were observed
between the NS1 proteins of each lineage (Table
6). A
total of 20 aa differences (7.1%)
were observed between the NS1
protein of lineage III and lineage IV,
while virus isolates from
1997 and 1998 of branch clusters IVi
and IVii differed by 3 aa
(1.1%). A fourth amino acid
substitution was observed at position
252 (S-P) in two of four virus
isolates of branch cluster IVi
(Nag98 and YN98) and three of four virus
isolates of branch cluster
IVii (Bei97, Shi5198, and Chi98). Amino acid
alignment of the
NS2 proteins revealed a conserved sequence with two
conserved
changes (1.6%) between lineages III and IV and only 1 aa
(0.8%)
difference between that of viruses from 1997 and 1998 of
sublineages
IVi and IVii.
Based on the evolutionary distances of the NS genes, the nucleotide
substitution rates for the NS genes of influenza B viruses
of lineages
III and IV were estimated to be 0.87 × 10
3 and
0.45 × 10
3 ns/st/yr, respectively. Although the NS
gene of lineage IV appeared
to be changing at a rate approximately two
times higher than that
of lineage III, both lineages were evolving at a
noticeably lower
rate than that of influenza A viruses (1.94 × 10
3 ns/st/yr) (
29). Similar variability was
subsequently observed
between substitution rates of the NS1 gene-coding
domain of lineages
III (1.03 × 10
3 ns/st/yr) and IV
(0.37 × 10
3 ns/st/yr). The NS2 coding region
demonstrated the lowest rate
of change since both lineages were
estimated to be evolving at
a rate of approximately 0.30 × 10
3 ns/st/yr. Similarly, amino acid substitution rates of
the NS1
proteins of lineage III and IV were quite different from one
another
but markedly lower than that of human H3N2 viruses (3.6 × 10
3 aas/st/yr) (
25). The NS2 protein of
influenza B viruses revealed
conservative substitution rates of
0.76 × 10
3 (lineage III) and 0.52 × 10
3 (lineage IV) aas/st/yr which were comparable to that
of the NS2
protein of human H3N2 viruses (0.50 × 10
3 aas/st/yr) (
25).
| |
DISCUSSION |
Comparison of the evolutionary profiles of the HA, NP, M, and NS
genes of influenza B viruses revealed that these genes of recent
isolates consistently divided into two distinct major lineages which
were evolving independently. Also, analysis of recent influenza B
viruses showed that viruses containing HA genes of both lineages cocirculated in Japan in 1998. It was also significant to reveal that
1998 viruses of lineage II demonstrated a novel amino acid deletion at
position 162 of the HA1 domain of the HA protein. This is yet another
example of the characterized systematic insertion and deletion of
nucleotides in the protein coding region of the HA protein of influenza
B viruses which potentially alters the antigenicity of these viruses
(20, 32, 39).
In spite of similar patterns of genetic divergence, the respective
evolutionary locations of the HA, NP, M, and NS genes of recent
influenza B viruses differed considerably. Figure
5 illustrates the proposed evolution of
influenza B viruses based on the phylogenetic characterization of the
HA, NP, M, and NS genes of viruses examined in this study. Following
phylogenetic divergence into two major lineages, genetic reassortment
apparently occurred among cocirculating viruses of each lineage, giving
rise to distinct viruses with novel genome constellations. It was
apparent that variable genetic constellations observed among influenza
B viruses were a reflection of frequent genetic reassortment among
cocirculating viruses. In fact, only six virus isolates (Sin79, Yam88,
Pan90, Mie93, Bei93, and Har94) contained genes which were consistently
located in the same respective lineage, while the remaining virus
isolates demonstrated reassortment of at least one gene segment. It was interesting to observe that divergent NP, M, and NS genes, represented by those of Nor84, were demonstrated to have been isolated
independently of the earliest divergent HA gene of lineage III,
represented by that of Iba85. Gene constellations of recent virus
isolates from 1997 and 1998 provided additional evidence of genetic
exchange among cocirculating viruses. While the HA genes of isolates
from 1997 and 1998 belonged to two distinct lineages with high amino acid variability, the internal NP, M, and NS genes of these viruses, on
the other hand, were less heterogeneous as they were included in
similar lineages. Recently, evidence for reassortment of genes coding
for the internal proteins of cocirculating human A H3N2 viruses was
reported (26), although these genes were demonstrated to
evolve essentially in a single major lineage. Observed reassortment among variable cocirculating internal genes of influenza B viruses was
rather more similar to characterized reassortment among genetically variable influenza C viruses (6, 34). However, in contrast to that of influenza A and C viruses, reassortment among viruses of
distinct lineages of influenza B viruses resulted in more pronounced protein variability.
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|
FIG. 5.
Proposed pattern for evolution and gene reassortment
among influenza B viruses. Genome constellations of influenza B viruses
are represented as follows. The HA genes of lineages II and III are
represented by blue diamonds and red diamonds, respectively; NP genes
of lineages of II and III are represented by blue squares and red
squares, respectively; M genes of lineages II and III are represented
by blue circles and red circles, respectively; the corresponding
lineages of NS genes (lineages III and IV) are symbolized by blue
triangles and red triangles, respectively.
|
|
It was further revealed that genes of a particular genetic lineage of
the internal genes may be sequestered for a period of time and, through
genetic reassortment, reemerge to circulate in later viruses. This
pattern was observed in lineage III of the evolutionary tree of the M
gene as well as lineage IV of the NS gene. As shown in Fig. 5, genes of
NS lineage IV first appeared in 1984, after which time genes of this
lineage were not observed in circulating viruses for a period of 9 years. However, as represented by the NS gene of Gua93, genes of this
lineage reemerged in 1993 and subsequently circulated in viruses from
1994, 1997, and 1998. Conversely, genes of lineage III were found in
most isolates from the 1980s and early 1990s but were not observed in
viruses isolated after 1994 (Har94) examined in this report. In a
similar fashion, M genes of lineage III reappeared in 1993 (Gua93)
after a 5-year hiatus (Aic88) but were not observed in later isolates.
Sequestration and reemergence of genetic lineages through genetic
exchange among circulating B viruses make prediction of genomic
constellations of future influenza B viruses very difficult, if not
impractical. Nonetheless, the question as to how these lineages are
being sequestered is raised. Possible suggestions include subclinical
infections by influenza B viruses which would not be detected, as well
as circulation of influenza B viruses in an as-yet-undetermined host. However, because sequence data for the internal genes of these viruses
is quite limited, the possibility that viruses containing genes of
sequestered lineages have simply not been analyzed must also be considered.
Essentially identical topologies of the phylogenetic trees of the M and
NP genes are noteworthy as they are typical of genes which are
physically linked, suggesting a possible association between the M and
NP proteins. The deduced M1 proteins of genes of both lineages,
however, showed no conserved amino acid differences, while the BM2
protein unexpectedly demonstrated exceptionally high amino acid
variability which was actually higher than that of the HA protein.
Therefore, it was not only interesting to observe that the most
variable (BM2) and least variable (M1) proteins were encoded on the
same genome segment but that similar evolutionary patterns of the M and
NP gene segments would more likely be the result of a codependent
association between the NP and BM2 proteins than between the NP and M1
proteins. Further investigation into the role of the BM2 protein
regarding its possible association with the NP protein would be warranted.
The evolutionary rates of the HA, NP, M, and NS genes of influenza B
viruses were determined to be generally lower than those of their
influenza A virus counterparts. Despite this fact, influenza B viruses
were found to employ a series of evolutionary mechanisms which
contributed to the variability of these viruses, including (i) genetic
divergence of genes coding for the surface HA protein and internal
proteins into two distinct major lineages, (ii) cocirculation of
multiple lineages for extended periods of time, (iii) frequent reassortment among circulating viruses giving rise to new variants with
distinct genome constellations, (iv) sequestration and reemergence of
gene lineages, and (v) systematic deletion and insertion of nucleotides
in the HA gene. Thus, despite the lower evolutionary rates of influenza
B viruses, it is proposed that, through employment of these mechanisms,
new variants of influenza B viruses with distinct genome constellations
are generated, resulting in seasonal epidemic episodes. Furthermore,
such complex phylogenetic patterns may be indicative of a symbiotic
relationship among divergent viruses where timely exchange of gene
segments provides the variability necessary for these viruses to
continue circulating. This report demonstrates the fundamental
differences between the evolutionary mechanisms of human influenza A
and B viruses. Through ongoing global surveillance of influenza
viruses, and genetic analysis of the genes coding not only for the
surface HA protein but also for the internal proteins of these viruses,
we can better understand the evolutionary mechanisms of these viruses.
Further analysis of the remaining genes of influenza B viruses (NA, PA,
PB1, and PB2 genes) would be worthwhile to better characterize the
phylogenetic and reassortment patterns of these viruses.
| |
ACKNOWLEDGMENTS |
This study was undertaken in collaboration with local prefectural
institutions of hygiene in Japan. We are grateful to all staff members
working in prefectural institutions of hygiene for their excellent
surveillance activities.
This research was supported by research grants provided by the Japanese
Ministry of Health and Welfare.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology I, National Institute of Infectious Diseases, 23-1, Toyama
1-chome, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: (03) 5285-1111. Fax: (03) 5285-1155. E-mail: knerome{at}nih.go.jp.
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Top
Abstract
Introduction
